ABSTRACT The behavior of stage IV American lobster (Homarus
americanus) larvae in relation to various water temperatures was
observed in laboratory. Time budgets were documented in 2009 in trials
using a single larva per experimental enclosure in relation to three
temperature exposures (10[degrees]C, 15[degrees]C, and 20[degrees]C).
Larvae acclimated at a given temperature (15[degrees]C and 20[degrees]C)
were used. The same treatments were used in 2010, where one larva was
observed within a group of five individuals per experimental enclosure.
Larvae took less time to hide when they were acclimated to 15[degrees]C
and then exposed to 20[degrees]C. Video observations showed that larvae
were stressed (repeated tail flicks) when treatments simulated important
heat shocks (e.g., larvae acclimated at 20[degrees]C and exposed to
10[degrees]C). Results for single larva were similar to those from
groups of larvae. The tail-flicks display was also observed in 2010 with
larvae acclimated at 15[degrees]C and then exposed to 10[degrees]C.
Observations from both years showed that the time budget was similar
regardless of the larval cohort used in trials (different brood stocks
and times of the season). Overall, results confirm that water
temperature affects the behavior of lobster larvae. Acclimation to a
given temperature, however, tends to show that larvae may acclimate to
cold water. This study provides interesting results to better understand
the ecology of American lobster, particularly at the time of settlement.
These results should also be of interest for the lobster industry, which
looks to improve stock enhancement procedures through, for instance,
lobster-stocking programs.

The American lobster (Homarus americanus) is a common species found
along the east coast of North America from Labrador to South Carolina
(Observatoire global du Saint-Laurent-OGSL 2012). In Canada, lobsters
are found in high abundance in the southern Gulf of St. Lawrence (sGSL)
as well as off the southwest coast of Nova Scotia. The life cycle of
lobster has a long-pelagic phase during which larvae are exposed to
currents that may affect, as they reach stage IV, their distribution
upon settlement. At that stage (competent stage), lobster larvae are
known to leave the pelagic habitat for the benthic habitat (Barshaw
& Rich 1997, James-Pirri & Cobb 2000, Paille et al. 2002). Once
on the bottom, larvae rapidly seek a shelter to limit their
vulnerability to predators (Botero & Atema 1982, Boudreau et al.
1990, Palma et al. 1998).

Marine invertebrates, including crustaceans, are affected by many
environmental parameters. The American lobster, for instance, is able to
detect minute temperature variations. Jury and Watson III (2000) showed
that adult lobsters could discriminate temperature oscillations as low
as 0.15[degrees]C, as shown by variations in heart rates. Water
temperature may affect lobster growth (Cobb et al. 1983, Boudreau et al.
1991), size or age at sexual maturity (Waddy & Aiken 1991), and
locomotion (Jury & Watson III 2000, Magnay et al. 2003). Temperature
may also affect lobster reproduction by modifying (1) mating period and
duration, (2) molt synchronization, (3) the egg production cycle and
attachment, (4) incubation success, and (5) larval release (Waddy &
Aiken 1991). In addition, larvae released from eggs that developed at
warm temperatures contain higher energy reserves than those released
from eggs at cold temperatures (Sasaki et al. 1986). In the natural
habitat, high larval survival rates are observed early during the summer
when water temperature increases rapidly (Ennis 1995).

The response of lobster larvae to temperature, including survival,
may vary in relation to their developmental stage. Except for stage II
larvae, stages I, III, and IV larvae are tolerant to high temperatures,
particularly when exposed for short periods (<6 h) (Gruffydd et al.
1975). MacKenzie (1988) observed that stages I and II larvae reared at
10[degrees]C had a mortality rate of 10% and 39%, respectively, compared
with those reared at 12[degrees]C (<1% and 13%, respectively). Stages
III and IV larvae had a high mortality rate (>70%) when reared at
10[degrees]C relative to those reared at 12[degrees]C. These results
suggest that mortality may be great when larvae develop under low
temperature conditions, particularly when individuals reach competency.

Low water temperature may also affect larval behavior as well.
Annis (2005) showed that only 2% of stage IV larvae swim to the bottom
at temperatures less than or equal to 12[degrees]C. This observation
supports results from MacKenzie (1988) on the occurrence of a
12[degrees]C thermal threshold for stage IV larvae. Water temperature
may also influence other behaviors of stage IV larvae, such as
exploration on the sea floor, shelter selection (cryptic behavior), and
the number of times individual larvae travel between the bottom and the
water column before settling. Any delays in settlement may increase the
vulnerability of larvae to pelagic and benthic predators (Botero &
Atema 1982).

The American lobster is one of the most important marine resources
in Canada. Revenues in 2008 exceeded CA$ 920 million (Peches &
Oceans Canada 2008). Though many fishery restrictions exist to protect
the lobster stocks, the number of lobster landed in the sGSL decreased
in the 1980s. Landings have since improved due to conservation measures
and favorable ecosystem conditions except in the Northumberland Strait
where they have continued to decline (Comeau et al. 2004, Department of
Fisheries and Oceans 2007, Comeau et al. 2008). In response to this
decline, the lobster industry initiated a lobster enhancement program to
increase stock abundances using current knowledge and technologies. The
literature shows that a period of 11 days is sufficient to reach stage
IV larvae at 22[degrees]C (Castro & Cobb 2005), as compared with 54
days at 10[degrees]C (Ennis 1995). Larvae obtained from hatcheries for
enhancement programs are usually reared at temperatures between
18[degrees]C and 20[degrees]C for cost-effectiveness. Stocking, however,
may sometimes be done under cold (10-12[degrees]C) field conditions and
it is suspected that thermal shocks may affect larval survival and
behavior, particularly their cryptic behavior. It is thus important to
document the relationship between temperature and lobster larval
behavior.

The objectives of this study were to: (1) describe the behavior of
stage IV lobster larvae in relation to relevant water temperatures; (2)
verify if a thermal shocks occurs such that larval behavior is impacted;
(3) verify if acclimation to a given temperature could minimize a
potential thermal shock; (4) verify if the behavioral response of larvae
at different temperatures is similar when observations are done on
single larvae as compared with larvae within a group of larvae (effect
of interactions between individuals). It is predicted that the general
behavior of lobster larvae will be affected by water temperature such
that larvae observed in a high temperature environment will (1) spend a
longer period hidden, (2) reach the substrate rapidly, and (3) have a
low level of stress. In the event that water temperature has an effect
on general larval behavior, acclimation at a given temperature should
minimize this effect. Larvae acclimated to a given temperature will
spend longer periods hidden, be faster to reach the substrate, and have
a lower level of stress. Results from this study should provide some
ecological information on the effect of water temperature on the
behavior of lobster larvae whereas swimming toward the bottom and
finding a shelter and settling, which will provide information to the
lobster industry to improve enhancement techniques.

MATERIALS AND METHODS

Rearing Conditions of Stage IV Larvae

Stage IV American lobster larvae were obtained from the Coastal
Zones Research Institute (CZRI) in Shippagan (NB), Canada. The CZRI used
berried females captured from various locations in the sGSL. Once
released, larvae were collected from holding tanks at the water surface
using a small mesh net and placed in 1,200 1 tanks at 20-22[degrees]C at
an initial density of 10 larvae/1. These tanks had a circular double
drain and were supplied with water at a rate of 5 1/min. An aeration
system produced an intense bubbling in the water column to obtain a
homogeneous distribution of food and to avoid cannibalism between
larvae. Larvae were fed a mixture of dried food (Salt Creek brine shrimp
flakes; Artemac) and frozen Artemia (Hikari, Kyorin Ltd.) daily. The
photoperiod used was 16 L:8 N h to simulate summer conditions for our
region (47[degrees]N). Once stage IV was reached (12 days after
hatching), individuals were immediately separated from the other stages
to avoid cannibalism. Larvae were then transferred by car from the
hatchery to the Universite de Moncton (Moncton, NB) for the experiments
(travel time of 3 h).

Behavioral Observations on Single Larvae

The experiments were done in a laboratory in July and August 2009
using only stage IV larvae. Once in Moncton, larvae were maintained in
four enclosures (50 X 40 X 40 cm height) filled with artificial seawater
(28%-30%) using a density of 100-120 larvae/enclosure. Larvae were
acclimated for a period of 4 days at two different temperatures:
15[degrees]C (experimental group) and 20[degrees]C (control). The
20[degrees]C temperature represents the temperature used in hatcheries
to rear larvae. The 15[degrees]C temperature represents an intermediate
water temperature that lobster larvae may experience during the summer
in their natural habitat. Photoperiod, diet, and feeding periods used
during the experiments were the same as those at CZRI. Once acclimated,
a larva was captured and placed in a treatment enclosure and exposed to
a given temperature (10[degrees]C, 15[degrees]C, and 20[degrees]C).
These temperatures may be observed in bottom waters during the summer in
the sGSL (Chasse et al. 2006, Comeau 2006). Each treatment was done in
triplicate for a total of 18 trials per day of observation (three
temperature exposures X two acclimation temperatures X three
replicates). Seven batches of stage IV larvae were used during the
summer for a total of 126 trials. These batches were used to quantify
changes in the larval behavior of lobsters that could be related to an
increase in water temperature during the summer period as well as to the
development of berried females in the hatchery.

Each treatment enclosure (as described above) contained a
sterilized 1-cm thick mixed gravel layer on the bottom and a shelter
made from three stones (ca 20 x 19 x 8 cm height). A small amount of
water (0.175 1) from the acclimation enclosures was added in the
treatment enclosure before the first trial to insure that the first
larva exposed to a treatment had the same chemical signals (congeneric
odors) as larvae used in the subsequent treatments within the same
enclosure. Water in the experimental enclosures was not changed between
trials for a given batch of larvae as larval behavior may be modified by
chemical signals (Moore et al. 1991).

[FIGURE 1 OMITTED]

For each trial, a larva was introduced to the enclosure using a
funnel attached to a plastic pipe to mimic the releasing technique used
by the industry during enhancement in the field. The larva was
videotaped following the release using a Sony Handycam DCR-SR47 over a 1
h period. Six treatment enclosures were used simultaneously with each
being videotaped independently. A total of 18 videos were filmed every
observation day. The 18 treatments were randomly assigned during the
day. All observations were done between 9 am and 4 pm. Videotapes were
subsequently viewed and analyzed to quantify time budgets (Table 1).

[FIGURE 2 OMITTED]

Behavioral Observations on Larvae Within a Group of Larvae

The experiment was repeated between July and August 2010 using
groups of five larvae. The goal of this experiment was to investigate
and describe the time budget of single larvae within groups of larvae in
enclosures. Although the experiments were similar to those done with
single larvae, a larger diameter pipe was used to release five larvae
simultaneously in the treatment enclosure. Because hatching occurred
earlier in 2010 than in 2009, observations on the last larval batch were
obtained for only two replicates per treatment (total n = 120).

Data Analysis

All videotapes were viewed to determine larval time budgets and
provide a general description of the behaviors displayed by stage IV
lobster larvae. Four main behaviors were observed: hiding, swimming,
exploring, and surfacing (swimming directly at the water surface).
Because multicollinearity was evident for time budget data, multivariate
analyses (e.g., MANOVA) using all behaviors within the same statistical
model were not done. The following dependent variables were analyzed in
2009 (one larva/enclosure): the percentage of time a larva was hiding,
the time used by the larva to reach the substrate, the number of times a
larva left the substrate or the shelter, and the duration of tail flicks
(proxy used a stress measure). The following dependent variables were
analyzed in 2010 (five larvae/enclosure): the time used by the first
three larvae (out of the five) to reach the substrate and the duration
of tail flicks. All analyses were done using three-way analysis of
variance (ANOVA) with larval batch, acclimation temperature, and
temperature exposure as independent variables. All data were
nonparametric rank transformed to meet the assumptions of parametric
analyses (Conover & Iman 1981), which were done using SAS 9.1. (SAS
Institute 2003).

RESULTS

Behavioral Observations on Single Larvae

Overall, the time stage IV lobster larvae spent hiding within a 1 h
time frame was high for all larval batches (Table 1; Fig. 1). Larvae
spent at least 60% of their time hiding except in a few scenarios
(acclimation at 20[degrees]C and exposed to 10[degrees]C in batches 1
and 2, acclimation at 15[degrees]C and exposed to 20[degrees]C in batch
5). Exploration was the second most important behavior displayed by
larvae (Table 1; Fig. 1). Larvae from all treatments and larval batches
spent less than 10% of their time swimming or surfacing. Hiding behavior
was investigated in further detail as it was the most important behavior
displayed by stage IV lobster larvae in terms of occurrence as well as
for its effect on survival (hidden larvae will be less vulnerable to
predators).

The percentage of time larvae spent hiding varied among larval
batches and with acclimation and exposure temperatures (Fig. 2). For
instance, the percentage of time spent hiding was often greater than 80%
but dropped to less than 60% in two particular larval batches when
larvae were acclimated at 20[degrees]C and exposed to 10[degrees]C. The
time larvae spent hiding varied with acclimation temperature and was
also a function of the interactions between acclimation temperature and
both larval batches and temperature exposure (Table 2).

The time that larvae took to reach the substrate was usually less
than 300 sec except for certain larval batches for which greater than
1,000 sec were needed (Fig. 3). Time to reach the substrate varied
significantly with temperature exposure and as a function of the
interaction between acclimation and exposure temperatures (Table 3). The
number of times larvae left the substrate was generally higher when
individuals were acclimated at 15[degrees]C compared with individuals
acclimated at 20[degrees] C (Fig. 4). The number of times larvae left
the substrate varied with acclimation temperature and as a function of
the interactions between both larval batches and temperature exposure
(Table 4).

[FIGURE 3 OMITTED]

Almost no tail flicks were observed for larvae acclimated at
15[degrees]C for all temperature exposures (Fig. 5). Larvae displayed
tail flicks very often when acclimated at 20[degrees]C and exposed to
10[degrees]C. Duration of tail flicks (multiple succeeding occurrences)
varied between 1 and 10 sec as a function of both acclimation and
exposure temperatures and as a function of the interaction between these
two variables (Table 5).

Behavioral Observations on Larvae Within Groups of Larvae

As observed for single larvae, the time budget for stage IV lobster
larvae within a group of five individuals showed that larvae spent most
of their time hiding (Fig. 6). The percentage of larvae hiding reached
almost 80% within the first 5 min for larvae acclimated at 15[degrees]C
for all temperature treatments. The percentage of larvae hiding was over
80% after 5 min of observation for larvae acclimated at 20[degrees]C,
except in treatments where larvae were exposed to 10[degrees]C. Under
that temperature treatment, it took 30 min for 80% of larvae to be
hidden. A relatively stable time budget was observed after this with a
constant proportion of time devoted to surfacing, swimming, or exploring
under all treatments. Overall, the percentage of larvae hiding was low
when temperature exposures were lower than the acclimation temperature
(acclimation at 15[degrees]C and exposed to 10[degrees]C, acclimation at
20[degrees]C and exposed to 10[degrees]C and 15[degrees]C). These data
could not be examined statistically.

The time taken by the first three larvae to reach the substrate was
generally fast (Fig. 7) but increased when the temperature exposure was
lower than the acclimation temperature (acclimation at 15[degrees]C and
exposed to 10[degrees]C, acclimation at 20[degrees]C and exposed to
10[degrees]C or 15[degrees]C). This period of time also varied among
larval batches and as a function of the acclimation temperature by
temperature exposure interaction (Table 6).

As for the single larva experiments, the tail-flick behavior was
not common for larvae acclimated at 15[degrees]C (Fig. 8). A few tail
flicks were observed when individuals were exposed to 10[degrees]C. The
tail-flick duration in these particular treatments was less than 5 sec.
Larvae displayed tail flicks often when acclimated at 20[degrees]C and
exposed to 10[degrees]C. The tail-flick duration varied between 10 and
40 sec within that treatment. The duration of tail flicks varied
significantly with the acclimation temperature by temperature exposure
interaction (Table 7).

DISCUSSION

The time budget of stage IV lobster (Homarus americanus) larvae
revealed that hiding was the most commonly displayed behavior by
individuals during the transition phase between the pelagic and benthic
stages. The hiding behavior was common for all treatments and larval
batches, confirming that stage IV larvae are competent and that they
will rapidly seek a refuge to decrease their vulnerability to predators
(Stein & Magnuson 1976, Barshaw & Rich 1997, Chau et al. 2009,
Jones & Shanks 2009). Exploration was the second most important
behavior. This is consistent with observations made by Karnofsky et al.
(1989) on American lobsters. Lobster larvae start to explore the
substratum for an appropriate shelter as soon as they reach stage IV.
The exploration behavior, however, was quite variable in this study and
tended to be related, although not tested statistically, with the larval
batches. Swimming and surfacing were not important in terms of absolute
time allocated to these behaviors. Transitory locomotory behaviors were
displayed to get from the top of the water to the bottom (swimming) or
from the bottom to the surface (surfacing). Transitory movements are
generally observed when bottom environmental conditions are not optimal
(e.g., absence of shelters, soft-bottom habitats) (Boudreau et al. 1990,
1993).

[FIGURE 4 OMITTED]

The hiding behavior showed a relatively high variability in all
treatments, as indicated by the significant three-factor interaction.
The variability within a larval batch was greater for larvae acclimated
at 15[degrees]C than those acclimated at 20[degrees]C. Larvae were
probably less stressed to explore and swim in a warm environment after
being acclimated at a low temperature. The variability associated with
larval batches could be related to female sizes, as larvae from early
summer come from larger females than those obtained late in the summer.
The size shift and physiological state of females throughout the season
may affect eggs and larval development. Sibert et al. (2004) showed that
there are seasonal variations in the lipid contents of American lobsters
eggs. These observations were also made yearly by Wickins et al. (1995)
for the European lobster (Homarus gammarus). The amount of lipids, such
as triglycerides, will play an important role in the energy content of
larvae and ultimately their activity level (Theriault & Pernet
2007). Stage IV larvae with high lipid contents may then get to the
bottom and explore for shelters more rapidly than larvae with low lipid
contents. This may also help larvae to better compete with conspecifics
(or individuals from other species) for shelter space and diminish their
vulnerability to predators.

The percentage of time allocated to hiding was high and varied
least among larvae acclimated at 20[degrees]C (except for two particular
batches of larvae exposed to 10[degrees]C). A larva developing at
20[degrees]C may age physiologically more rapidly than a larva
developing at 15[degrees]C even though they have the same age in terms
of the time passed since hatching. Larvae acclimated at 20[degrees]C may
thus be more physiologically advanced in their development and show a
higher level of competency than larvae acclimated at 15[degrees]C. A
similar relationship was observed in the laboratory and field by
McCormick and Molony (1995) in the tropical goatfish Upeneus tragula.
These authors suggested that small changes in water temperature
(30[degrees]C versus 25[degrees]C) may have the potential to influence
the developmental rate to metamorphosis of tropical reef fishes and
subsequently their settlement behavior.

Results from the video observations on larvae within a group of
larvae showed similar responses as those from single larvae in relation
to temperature exposures and acclimation. Overall, a reduction in the
time allocated to hiding was observed when the exposure water was warmer
than the acclimation temperature. Eighty percent of larvae acclimated at
15[degrees]C and exposed to 10[degrees]C took about 5 min to hide
compared with only 2 min when larvae were acclimated at 15[degrees]C and
exposed to 15[degrees]C and 20[degrees]C. The period of time to reach
the same hiding level (80% of larvae) increased when larvae were
acclimated at 20[degrees]C and exposed to 10[degrees]C or 15[degrees]C.
This period decreased for larvae acclimated at 20[degrees]C and exposed
to 20[degrees]C. These laboratory results suggest that lobster larvae
exposed to large temperature differences (5[degrees]C and 10[degrees]C)
may allocate more time for other behaviors (e.g., swimming, tail flicks)
and potentially increasing their vulnerability to predators under
natural settings.

Stage IV lobster larvae generally took little time to reach the
substrate, regardless of the acclimation and exposure temperatures and
larval batch. Patterns were generally the same for larvae acclimated at
15[degrees]C except for larval batches 6 and 7. Larvae acclimated at
20[degrees]C showed that larvae may have suffered a thermal shock when
exposed to 10[degrees]C. Low temperatures may stress larvae (display of
tail flicks) and increase their swimming or surfacing time.
Surprisingly, this was observed in only two of the seven larval batches
(first two batches). As discussed above, increase in swimming time will
increase the vulnerability of larvae to predators. Van der Meeren
(2000), for instance, found in the field that over 10% of reared larvae
were caught by predators within the hour following their introduction.
Larvae must reach the bottom quickly to maximize survival.

[FIGURE 5 OMITTED]

The number of times larvae left the substrate or shelter was
generally low for those acclimated at 20[degrees]C relative to those
acclimated at 15[degrees]C. This was also observed for larvae acclimated
at 20[degrees]C and exposed to 10[degrees]C. Larvae acclimated at
20[degrees]C may have developed greater competency than those acclimated
at 15[degrees]C. Larvae with high competency may display more stable
behaviors than do larvae with low competency and stay in their shelter
more effectively or dig into the gravel. This particular behavior may,
however, also be related to larvae being less active at low
temperatures, which may lead them to restrict their movement. Van der
Meeren (2000), for instance, showed that low temperatures tended to
reduce the aggressivity in European lobsters, which led individuals to
stay in their shelter for a longer period.

The display of tail flicks was greatest when the difference between
acclimated and exposure temperatures was high. This behavior was
particularly obvious when larvae were acclimated at 20[degrees]C and
exposed to 10[degrees]C. This stress response was likely induced by a
thermal shock. Larvae within larval groups from all acclimation
temperatures displayed great tail-flick behavior when the exposure
temperature was 10[degrees]C. Tail-flick duration and the number of
larvae that displayed this behavior was, however, greater for larvae
acclimated at 20[degrees]C than those acclimated at 15[degrees]C. In
most cases, the tail-flick behavior was displayed within a 5 sec time
frame for larvae acclimated at 15[degrees]C but was almost 40 sec for
larvae acclimated at 20[degrees]C. Observations made in this study
during the 10[degrees]C exposure treatments tend to confirm findings by
various authors. Larval development is known to be inhibited below
10[degrees]C (Waddy & Aiken 1998, Wahle & Fogarty 2006).
MacKenzie (1988) showed that stage IV larvae will not reach stage V at
temperatures less than 12[degrees]C. A temperature of 12[degrees]C may
be the minimum temperature at which larvae remain viable (MacKenzie
1988). Similarly, Annis (2005) found that stage IV larvae remained in
surface waters above 12[degrees]C and rarely descended toward bottom
waters below this temperature. This temperature appears to be a
threshold temperature for many biological features and support the
observations made in this study on stressed individuals. Van der Meeren
(1991) also observed this particular behavior in her work. Larvae tend
to respond to inadequate conditions by displaying tail flicks and
swimming high in the water column. This may increase their vulnerability
to predators because tail flicks may alert and attract predators.

[FIGURE 6 OMITTED]

CONCLUSION

Overall, the results presented in this study tend to support the
initial working hypotheses and predictions. Figure 9 summarizes the main
observations. Water temperature affects the behavior of stage IV
American lobster larvae. Acclimation to cold water temperatures
decreases the effect of a thermal shock suffered by larvae. Stage IV
lobster larvae hide faster in warm water than in cold water. Results
also show that lobster larvae may reach the bottom quickly in a cold
environment once acclimated at low temperatures. Acclimated lobster
larvae, however, may leave the substrate more often. This may increase
their vulnerability to predators.

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

This study provides interesting results to better understand the
ecology of the American lobster in a context where larval information is
being used to model dispersal and to better understand potential climate
change effects on settlement behavior. The results of this study should
also be of interest for the lobster industry. Acclimation could improve
enhancement in cold habitats and help increase coastal lobster stocks.
These results must, however, be validated by field studies that more
closely simulate the methodology and the natural settings in which
enhancement programs are carried out. Enhancement techniques would be
improved if they decrease the amount of time larvae spent in the water
column and increase their time in natural shelters after seeding. This
must be done by reducing larval stress. Tail flicks will increase the
time spent in the water column and ultimately contacts with predators.

[FIGURE 9 OMITTED]

ACKNOWLEDGMENTS

We thank Remy Hache and his team from CZRI for their work during
the production of lobster larvae in 2009 and 2010. We also thank Yves
Hebert for his technical contributions. Michel Comeau and Stephan Reebs
were instrumental in providing equipment and advices during the project.
Additional thanks to Fabrice Pernet and Chris McKindsey for reviewing
the manuscript. Funding for this research has been provided by grants
from Homarus Inc., Fisheries and Oceans Canada, Canadian Foundation for
Innovation, Canadian Capture Fisheries Research Network (a strategic
network funded by NSERC) and Universite de Moncton.